Relatively short polynucleotide sequences are commonly used to selectively hybridize to a target polynucleotide sequence for various reasons. One such example is polymerase chain reaction (PCR) primers, which are typically designed in pairs to specifically amplify the sequence between them on a target polynucleotide. Like primers, a probe, whether used in real time PCR, in situ hybridization or on microarrays, can be used to identify the presence or concentration of the target sequence. Another example is a small interfering (siRNA), which mediates RNA interference that selectively silences a target gene. For the purpose of this disclosure, a polynucleotide that is designed to selectively hybridize or bind to a target polynucleotide is generically referred to as a “polynucleotide probe” or simply a “probe.” In this context, the fragment of the target polynucleotide that actually forms hydrogen bonds with the probe is referred to as the “target fragment.”
Various factors contribute to the ability of a probe to selectively and effectively bind a target fragment on a target polynucleotide. For instance, off-target DNA or RNA sequences that bear certain sequence identity to the probe sequence can bind to the probe and thus interfere with the probe's ability to bind the target polynucleotide. Likewise, secondary structure of an RNA molecule that involves binding between the target fragment and another fragment in the RNA molecule will require disruption energy to free the target fragment from such binding in order to make the target fragment accessible for binding to the probe. Determination of the secondary structure of an RNA molecule, however, is difficult.